A detailed understanding of how common oncogenic signaling pathways are assembled into larger signaling networks is essential to developing therapeutic strategies to properly target these pathways in cancer and for interpreting clinical outcomes from targeted therapeutics. While the effected oncogenes and tumor suppressors that predominate different classes of human cancer can vary greatly, a small number of highly integrated signaling nodes are affected in the majority of human cancers, regardless of tissue of origin. It is important to understand how these key signaling nodes are regulated and what the downstream consequences are for tumor development, progression, and treatment. In this project, we focus on one such node, involving the TSC1-TSC2 complex and the Ras-related small G protein Rheb, which is aberrantly regulated in nearly all genetic tumor syndromes and the most common forms of sporadic cancer. Currently, the only known downstream target of this small G protein switch is the mammalian target of rapamycin (mTOR). The aims of this project will employ both hypothesis-driven approaches, based on studies from the first 4 years of this P01, and unbiased genomic and proteomic screens. The aims are designed to 1) reveal new components, connections, and dowstream targets within the TSC-Rheb signaling network, 2) identify and characterize previously unexplored therapeutic strategies to target this network in tumors, 3) identify novel biomarkers to predict and monitor therapeutic responses, 4) serve as a discovery-based platform to fuel the preclinical elements in projects 2 and 3 of this program, and 5) bioinformatically analyze and integrate the large cross-species data sets generated within all projects of the program. To achieve these goals, we will closely integrate high-throughput technologies in Drosophila (Perrimon laboratory) with mechanistic characterization and validation in mammalian cell and tumor models (Manning laboratory